Cloning of flavonoid 3′,5′-hydroxylase and 3′-hydroxylase homologs from black currant (Ribes nigrum) and their differential expression at various fruit maturation stages

Abstract

Flavonoid 3′-hydroxylase (F3′H) and 3′,5′-hydroxylase (F3′5′H) generate precursor molecules for the synthesis of cyanidin-based anthocyanins (red) and delphinidin-based anthocyanins (blue to purple) in Ribes nigrum L. (black currant). In this study, full-length 1780 and 1650-bp cDNA homologs of RnF3′5′H1 and RnF3′H1 from black currant were identified and cloned using a homologous cloning technique. Data revealed that Rn F3′5′H1 and RnF3′H1 are homologs that encode enzymes involved in anthocyanin synthesis from different plants, which phylogenetically cluster with the CYP75B and CYP75A families in the P450 superfamily, respectively. The enzymes encoded by these two genes also shared a high homology with flavonoid hydroxylases identified from other plants. Furthermore, RnF3′5′H1 and RnF3′H1 levels were upregulated during fruit maturation. RnF3′5′H1 levels were associated with both anthocyanin and soluble carbohydrate levels in blackcurrant, while RnF3′H1 expression did not have such an association. The structure and expression patterns of RnF3′5′H1 and RnF3′H1 in blackcurrant were also characterized. Further studies should aid understanding of anthocyanin biosynthesis in black currant to develop molecular approaches and manipulate anthocyanin production in blackcurrant.

Keywords

Anthocyanin / Flavonoid / Gene cloning / Expression / Blackcurrant

References

[1] Bogs J, Ebadi A, McDavid D, Robinson SP. Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development. Plant Physiol, 2006, 140(1): 279-291.

[2] Bohm BA. Introduction to Flavonoids, 1998, Amsterdam: Harwood Academic Publishers 339 364

[3] Brugliera F, Barri-Rewell G, Holton TA, Mason JG. Isolation and characterization of a flavonoid 3′-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida. Plant J, 1999, 19(4): 441-451.

[4] Castellarin SD, Matthews MA, Di Gaspero G, Gambetta GA. Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta, 2007, 227(1): 101-112.

[5] Chen Y, Mao Y, Liu H, Yu F, Li S, Yin T. Transcriptome analysis of differentially expressed genes relevant to variegation in peach flowers. PLoS ONE, 2014 9 6 e90842

[6] Czemmel S, Heppel SC, Bogs J. R2R3 MYB transcription factors: key regulators of the flavonoid biosynthetic pathway in grapevine. Protoplasma, 2012, 249(Suppl 2): S109-S118.

[7] Diaconeasa Z, Leopold L, Rugină D, Ayvaz H, Socaciu C. Antiproliferative and antioxidant properties of anthocyanin rich extracts from blueberry and blackcurrant juice. Int J Mol Sci, 2015, 16: 2352-2365.

[8] Forkmann G, Ruhnau B. Distinct substrate specific of dihydroflavonol-4-reductase from flower of Petunia hybrida. Plant Mol Biol, 1987, 13: 491-502.

[9] Graham SE, Peterson JA. How similar are P450 s and what can their differences teach us?. Arch Biochem Biophys, 1999, 369(1): 24-29.

[10] Grotewold E. The genetics and biochemistry of floral pigments. Annu Rev Plant Biol, 2006, 57: 761-780.

[11] Halbwirth H. The creation and physiological relevance of divergent hydroxylation patterns in the flavonoid pathway. Int J Mol Sci, 2010, 11(2): 595-621.

[12] Halliwell B, Rafter J, Jenner A. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not?. Am J Clin Nutr, 2005, 81(1 Suppl): 268s-276s.

[13] Han Y, Vimolmangkang S, Soria-Guerra RE, Rosales-Mendoza S, Zheng D, Lygin AV, Korban SS. Ectopic expression of apple F3′H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under nitrogen stress. Plant Physiol, 2010, 153(2): 806-820.

[14] Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry, 2000, 55(6): 481-504.

[15] Holton TA, Brugliera F, Lester DR, Tanaka Y, Hyland CD, Menting JG, Lu CY, Farcy E, Stevenson TW, Cornish EC. Cloning and expression of cytochrome P450 genes controlling flower colour. Nature, 1993, 366(6452): 276-279.

[16] Jeong ST, Goto-Yamamoto N, Hashizume K, Esaka M. Expression of the flavonoid 3′ hydroxylase and flavonoid-3′5′-hydroxylase genes and flavonoid composition in grape (Vitis vinfiera). Plant Sci (Amsterdam, Netherlands), 2006, 170(1): 61-69.

[17] Koes R, Verweij W, Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci, 2005, 10(5): 236-242.

[18] Koponen JM, Happonen AM, Mattila PH, Torronen AR. Contents of anthocyanins and ellagitannins in selected foods consumed in Finland. J Agric Food Chem, 2007, 55(4): 1612-1619.

[19] Lou Q, Liu Y, Qi Y, Jiao S, Tian F, Jiang L, Wang Y. Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth. J Exp Bot, 2014, 65(12): 3157-3164.

[20] Maatta-Riihinen KR, Kamal-Eldin A, MattilaPH Gonzalez-Paramas AM, Torronen AR. Distribution and contents of phenolic compounds in eighteen Scandinavian berry species. J Agric Food Chem, 2004, 52(14): 4477-4486.

[21] Mazza G, Miniati E. Anthocyanins in fruits, vegetables, and grains, 1993, Boca Raton: CRC Press p362

[22] Nakatsuka T, Nishihara M, Mishiba K, Yamamura S. Temporal expression of flavonoid biosynthesis-related genes regulates flower pigmentation in gentian plants. Plant Sci, 2005, 168(5): 1309-1318.

[23] Olsen KM, Hehn A, Jugde H, Slimestad R, Larbat R, Bourgaud F, Lillo C. Identification and characterisation of CYP75A31, a new flavonoid 3′5′-hydroxylase, isolated from Solanum lycopersicum. BMC Plant Biol, 2010, 10: 21.

[24] Pascual-Teresa SD, Sanchez-Ballesta MT. Anthocyanins: from plant to health. Phytochem Rev, 2008, 7: 281-299.

[25] Pirie A, Mullins MG. Changes in anthocyanin and phenolics content of grapevine leaf and fruit tissues treated with sucrose, nitrate, and abscisic Acid. Plant Physiol, 1976, 58(4): 468-472.

[26] Quattrocchio F, Wing JF, Leppen H, Mol J, Koes RE. Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell, 1993, 5(11): 1497-1512.

[27] Saito K, Yonekura-Sakakibara K, Nakabayashi R, Higashi Y, Yamazaki M, Tohge T, Fernie AR. The flavonoid biosynthetic pathway in Arabidopsis: structural and genetic diversity. Plant Physiol Biochem, 2013, 72: 21-34.

[28] Sakakibara H, Honda Y, Nakagawa S, Ashida H, Kanazawa K. Simultaneous determination of all polyphenols in vegetables, fruits, and teas. J Agric Food Chem, 2003, 51(3): 571-581.

[29] Schoenbohm C, Martens S, Eder C, Forkmann G, Weisshaar B. Identification of the Arabidopsis thaliana flavonoid 3′-hydroxylase gene and functional expression of the encoded P450 enzyme. Biol Chem, 2000, 381(8): 749-753.

[30] Tanaka Y, Brugliera F. Flower colour and cytochromes P450. Philos Trans R Soc Lond B Biol Sci, 2013, 368(1612): 1-14.

[31] Tanaka Y, Ohmiya A. Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways. Curr Opin Biotechnol, 2008, 19(2): 190-197.

[32] Tanaka Y, Yonekura K, Fukuchi-Mizutani M, Fukui Y, Fujiwara H, Ashikari T, Kusumi T. Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana triflora. Plant Cell Physiol, 1996, 37(5): 711-716.

[33] Taylor B, Powell A. Isolation of plant DNA and RNA. Focus, 1982, 4: 4-6.

[34] Toda K, Akasaka M, Dubouzet EG, Kawasaki S, Takahashi R. Structure of flavonoid 3′-hydroxylase gene for pubescence color in soybean. Crop Sci, 2005, 45(6): 2212-2217.

[35] Uimari A, Strommer J. Anthocyanin regulatory mutations in pea: effects on gene expression and complementation by R-like genes of maize. Mol Gen Genet, 1998, 257(2): 198-204.

[36] Wang J, Ming F, Han Y, Shen D. Flavonoid-3′,5′-hydroxylase from Phalaenopsis: a novel member of cytochrome P450 s, its cDNA cloning, endogenous expression and molecular modeling. Biotechnol Lett, 2006, 28(5): 327-334.

[37] Werck-Reichhart D, Feyereisen R. Cytochromes P450: a success story. Genome Biol, 2000, 1(6): 3003-1-3003.9.

[38] Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol, 2001, 126(2): 485-493.

[39] Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J Agric Food Chem, 2006, 54(11): 4069-4075.

[40] Xia X, Xie Z. DAMBE: data analysis in molecular biology and evolution. J Hered, 2001, 92: 371-373.

[41] Xu B, Li J, Zhang X. Cloning and molecular characterization of a functional flavonoid 3 of Petunia hybrida. Journal Physiol, 2007, 164(3): 350-363.